We have identified a mouse gene encoding a 65-kDa protein (FKBP65) that shares sequence homology with members of the FK506-binding protein (FKBP) family. FKBP65 has been shown to possess peptidyl-prolyl cis trans isomerase activity, a characteristic shared by other FKBP family members. Using a polyclonal antibody raised against the C-terminus, we have shown that FKBP65 co-precipitates the heat shock protein hsp90, and the serine threonine kinase c-Raf-1. Through the use of in vitro and in vivo cell culture systems we have demonstrated that FKBP65 preferentially binds to an "activated" form of c-Raf-1. We have used the rat pheochromocytoma cell line (PC12) as an in vivo cell system to study how FKBP65 overexpression influences signaling proteins within the ras- raf dependent pathway. Based on the ability to respond to both mitogenic (EGF) and differentiative (NGF) stimuli, PC12 cells have been used to identify and characterize many of the molecules that constitute this pathway. Parental cells and FKBP65 overexpressing cells were stimulated with either NGF or EGF for 48 hours. With NGF, clones overexpressing FKBP65 showed neurite outgrowth more rapidly than control PC12 cells. When cells were stimulated with EGF, unlike control cells, overexpressors produced neurite outgrowth similar to those observed following NGF stimulation. Further analysis showed that EGF stimulation resulted in a dramatic shift in the activation kinetics of MAP kinase, leading to prolonged activation relative to that observed with EGF stimulation of parental PC12 cells. These results suggest a role for FKBP65 in regulating the activity of signaling proteins within the ras pathway, an effect that has been proposed to discriminate between mitogenic and differentiation-specific outcomes of receptor stimulation.